Project Summary/Abstract Therapeutic development in a broad spectrum of diseases often involves drugs that target G protein- coupled receptors (GPCRs). However, despite the importance of GPCRs in disease pathogenesis or progression, receptor-targeted drugs often have surprisingly limited therapeutic effect. One reason is that multiple GPCRs with redundant functions drive disease pathogenesis or progression, as occurs in Alzheimer's, inflammatory disorders and many cancers. Thus, effective therapy would require concurrent targeting of multiple GPCRs, which often cannot be achieved because drugs targeting certain GPCRs do not yet exist, or all disease-driving GPCRs have yet to be identified. In other diseases, including uveal melanoma, hormone- secreting pituitary tumors and ~10-15% of all cancers, pathogenesis is driven independently of GPCRs by constitutively active mutant G protein ?-subunits. Here, GPCR-targeted drugs are inappropriate because they cannot prevent activation of mutant G proteins. However, both types of therapeutic roadblocks could be overcome by pharmacologically targeting G proteins instead of GPCRs. In addition to their clinical/translational potential, pharmacological agents that directly target specific G proteins would be extremely valuable as probes in basic science. They would provide simple, fast, cheap and reliable tools to identify novel functions of G proteins in normal physiology and in animal models of many diseases, in contrast to conventional knockout or knockdown strategies, which are slow, expensive, or suffer from compensatory or off-target effects. Furthermore, understanding how pharmacological agents inhibit specific G proteins will reveal fundamentally new mechanistic principles that control G protein activity. Accordingly, this project aims over the long term to develop a panel of pharmacological agents, each of which directly and selectively inhibits specific G protein ?-subunit subtypes, and describe their mechanisms of action in detail. Its foundation is a pair of nearly identical cyclic depsipeptide natural products that are bioavailable, potent and highly selective inhibitors of the Gq/11 subfamily of G protein alpha-subunits. Using a combination of synthetic organic chemistry, computational biology and G protein functional assays, the project team will pursue Specific Aims that will: 1) determine how these molecules inhibit Galpha activation; 2) identify features of these inhibitors that determine potency, pseudo-irreversibility and Galpha selectivity; and 3) identify synthetic analogs of these inhibitors that target constitutively active Gq/11. These Aims are founded on preliminary data showing that: 1) the project team has established the first robust, scalable route for synthesizing analogs of these inhibitors, as required for preclinical and, eventually, clinical studies; 2) the naturally produced inhibitor potently targets cells driven by constitutively active mutant Gq/11; and 3) the naturally produced inhibitor works by a mechanism that can be adapted to every Galpha subtype.